Fluorescently Activated Cell Sorting Followed by Microarray Profiling of Helper T Cell Subtypes from Human Peripheral Blood

<div><p>Background</p><p>Peripheral blood samples have been subjected to comprehensive gene expression profiling to identify biomarkers for a wide range of diseases. However, blood samples include red blood cells, white blood cells, and platelets. White blood cells comprise polymorphonuclear leukocytes, monocytes, and various types of lymphocytes. Blood is not distinguishable, irrespective of whether the expression profiles reflect alterations in (a) gene expression patterns in each cell type or (b) the proportion of cell types in blood. CD4⁺ Th cells are classified into two functionally distinct subclasses, namely Th1 and Th2 cells, on the basis of the unique characteristics of their secreted cytokines and their roles in the immune system. Th1 and Th2 cells play an important role not only in the pathogenesis of human inflammatory, allergic, and autoimmune diseases, but also in diseases that are not considered to be immune or inflammatory disorders. However, analyses of minor cellular components such as CD4⁺ cell subpopulations have not been performed, partly because of the limited number of these cells in collected samples.</p><p>Methodology/Principal Findings</p><p>We describe fluorescently activated cell sorting followed by microarray (FACS–array) technology as a useful experimental strategy for characterizing the expression profiles of specific immune cells in the circulation. We performed reproducible gene expression profiling of Th1 and Th2, respectively. Our data suggest that this procedure provides reliable information on the gene expression profiles of certain small immune cell populations. Moreover, our data suggest that GZMK, GZMH, EOMES, IGFBP3, and STOM may be novel markers for distinguishing Th1 cells from Th2 cells, whereas IL17RB and CNTNAP1 can be Th2-specific markers.</p><p>Conclusions/Significance</p><p>Our approach may help in identifying aberrations and novel therapeutic or diagnostic targets for diseases that affect Th1 or Th2 responses and elucidating the involvement of a subpopulation of immune cells in some diseases.</p></div

Schematic of the FACS–array procedure for peripheral blood cells.

<p>(<b>A</b>) Schematic of analysis of helper T (Th) cells using the FACS–array procedure for peripheral blood cells, where PBMCs are isolated on a density gradient centrifuge and stained with fluorescence-labeled antibodies (CD4-FITC, CXCR3-APC, and CCR4-PE) and PI for FACS. Total RNA was extracted from each subclass of lymphocytes and subjected to two rounds of cRNA amplification. Synthesized aminoallyl-aRNA samples were labeled with biotin and subjected to microarray analysis. (<b>B</b>) Dot plot imaging of FACS isolation of Th1 and Th2 cells. (1) Lymphocytic subpopulation of PBMCs was selected on the basis of their unique forward and side scatter properties on fluorocytometry. (<b>2</b>) Among the lymphocytes, PI-negative (viable) CD4⁺ Th cells were selected. (3) On the basis of the signal intensity of fluorostaining for the CXCR3 and CCR4 markers, the viable Th cells were separated into two subgroups: CXCR3⁺/CCR4⁻ as Th1 cells and CXCR3⁻/CCR4⁺ as Th2 cells.</p

Differences in expression levels of representative genes between human Th1 and Th2 cells.

<p>Gene expression profiles of human Th1 and Th2 cells isolated from blood samples of 12 healthy individuals were analyzed using Illumina Human-6v2 Expression BeadChips arrays. Each bar represents a fold change of averaged signal intensity each gene in the Th1 microarray data divided by averaged signal intensity of the same gene in Th2 microarray data. Indicated next to each bar diagram is the <i>p</i> value obtained from a paired <i>t</i> test to evaluate a difference in signal intensity for each gene between the Th1 and Th2 microarray data from the 12 donors. Colors indicate the signal intensity of genes. Red: a very high expression level (>5000), orange: high level of expression (1000–5000), yellow: medium expression level (200–1000), and white: low expression level (<200). (<b>A</b>) Fold changes of well-established Th1 and Th2 genes in the comparison between human Th1 and Th2 microarray data. Positive fold changes mean that transcripts are more abundant in Th1 microarray data than in Th2 data, and negative values indicate the opposite. (<b>B</b>) Fold changes of the most prominent Th1-specific genes, which showed a signal intensity of>200 and a fold change of>2 in Th1 microarray data compared with that in Th2 data. (<b>C</b>) Fold changes of the most prominent Th2-specific genes, which showed a signal intensity of>200 and a fold change of>2 in Th2 microarray data compared with that in Th1 data.</p

Evaluation of RNA amplification systems for the FACS-array procedure.

<p>(<b>A</b>) A summary of principles of the four RNA amplification systems and efficacy of amplification. Some data are shown as mean ± SD. (<b>B</b>) Scatter plots showing correlations between the gene expression profiles of duplicated batches of amplified RNA from the same small amount of human lymphocyte RNA using three RNA amplification systems. TotalPrep: amplified using the Total Prep RNA amplification kit (Illumina), Target Amp: amplified using the TargetAmp 2-round aminoallyl-aRNA amplification kit 1.0, and WT-Ovation: amplified using the WT-Ovation FFRE RNA amplification system V2 (NuGEN). (<b>C</b>) Scatter plots showing correlations between the gene expression profiles of amplified RNA from the same small amount of human lymphocyte RNA using TargetAmp/WT-Ovation and conventional TotalPrep.</p

Immunophenotype and clonality of the MLL-AF10/K-ras-induced leukemia.

<p>(A) Frequencies of GFP⁺/Venus⁺ cells or human CD45⁺ cells in the BM, spleen, and liver at 8 weeks after transplantation with human HSCs co-transfected with the MLL-AF10 and K-ras^G12V genes were examined by flowcytometric analysis. The flowcytometry data shown are representative of 6 to 8 mice per group in one representative experiment of two (left). The average of %frequencies of the GFP⁺ and Venus⁺ cells in whole cells in the indicated organs is shown with the standard deviation (right, upper; n = 6). The absolute cell number of human CD45⁺ cells in the indicated organs is shown with the standard deviation (right, lower; n = 6). (B) Representative RT-PCR results confirming the stable, long-term expression of the MLL-AF10 and Flag-K-ras^G12V transcripts in human hematopoietic cells in the BM of mice 8 weeks after transplantation. (C) Lineage distribution of the GFP⁺ and Venus⁺ cells in the BM of a mouse engrafted with HSCs expressing MLL-AF10 and activated K-ras. (D) Southern blot analysis of DNA prepared from the human blood cells in the spleen of mice receiving transplants of MLL-AF10/K-ras^G12V co-transduced HSCs. Independent leukemia samples derived from two mice (lane 1; mouse 1 and lane 2; mouse 2) were examined. DNA was digested with Bgl II and probed with an EGFP probe. M: marker.</p

Co-transduction of activated K-ras and MLL-AF10 into CD34⁺HSCs.

<p>(A) Schematic structure of the MLL-AF10-GFP and Flag-K-ras^G12V-Venus vectors. (B) Infectious efficiency of the MLL-AF10-GFP and Flag-K-ras^G12V-Venus co-transfection. The data and the summary shown in the flowcytometric analysis is representative of the transduced CD34⁺ HSCs in 2 experiments.</p

Flowcytometric analysis confirming multilineage engraftment.

<p>(A) Representative flowcytometric results of EV- or MLL-AF10-transduced human hematopoietic cells. The human CD45⁺ GFP⁺ cells were analyzed for their lineage distributions to B cells (CD19⁺), T cells (CD3⁺), and myeloid cells (CD33⁺). (B) Multilineage differentiation of MLL-AF10-transduced cells. The data shows cells gated on the CD45⁺GFP⁺ cell population. The graph represents the mean ± SD of the frequencies of CD33⁺ myeloid cells, CD19⁺ B cells, and CD3⁺ T cells in the BM (upper) and spleens (lower) of mice engrafted with EV-transduced (n = 8) or MLL-AF10-transduced (n = 6) CD34⁺ HSCs. No difference in the graft composition between the EV- and MLL-AF10-expressing CD34⁺ HSCs was found. Similar results were obtained in 3 independent experiments.</p

Enforced expression of MLL-AF10 augmented multilineage hematopoiesis, but was insufficient to induce leukemogenesis in vivo.

<p>(A) Representative RT-PCR results confirming the long-term expression of the MLL-AF10 transcript in the BM cells of mice 25 weeks after transplantation (lane 1; water, lane 2; cells from a mouse in the EV-transfused group, lane 3; cells from a mouse in the MLL-AF10-transfused group, and lane 4; positive control (MLL-AF10 plasmid)). (B) Flowcytometric analysis of the frequency of GFP⁺ cells. The indicated vector (EV, left or MLL-AF10, right)-transduced human CD34⁺ cells, whose <i>in vitro</i> GFP expression is shown in the upper panels (Before) of the flowcytometric analysis, were transplanted into NOG mice. Twenty-five weeks later, the GFP-expressing cells gated on human CD45⁺ hematopoietic cells in the BM was measured (lower panels of the FACS profiles). The data shown are representative of 3 independent experiments. The graphs show the frequency of GFP⁺ cells in human CD34⁺ cells just before transplantation (Before) and the mean ± SD of the frequency of GFP⁺ cells in the BM and spleen of mice receiving transplants of EV-transduced HSCs (n = 8) or of MLL-AF10-transduced HSCs (n = 6) 25 weeks after transplantation, in one representative experiment of three. Similar results were obtained in the 3 independent experiments.</p

Pathological phenotypes of the leukemia.

<p>(A) Hematoxylin and eosin staining showing the infiltration of leukemic cells in the indicated organs of mice engrafted with HSCs expressing the MLL-AF10 and K-ras^G12V genes compared to control mice. (B) Immunostaining by a human CD45 mAb in the BM, spleen, and liver in mice engrafted with HSCs expressing the MLL-AF10 and K-ras^G12V genes.</p

Cooperation of MLL-AF10 with activated K-ras induced acute monoblastic leukemia.

<p>(A) Kaplan-Meier survival analysis of mice receiving transplants of human HSCs transfected with EV (n = 8), K-ras^G12V (n = 12), MLL-AF10 (n = 6), or MLL-AF10 plus K-ras^G12V (n = 6) vectors. (B) GFP and Venus expression in peripheral blood cells at the indicated weeks after transplantation with human HSCs co-transfected with the MLL-AF10 and K-ras^G12V genes. (C) May-Giemsa staining of the peripheral blood of mice engrafted with human HSCs co-transfected with the MLL-AF10 and K-ras^G12V genes. Morphologic leukemia cells were found in the peripheral blood of these mice 50 days after transplantation. (D) Splenomegaly in the MLL-AF10/K-ras^G12V mice. Spleens from mice engrafted with EV-transduced HSCs (left) and MLL-AF10/K-ras^G12V co-transduced HSCs (right) are shown. The graph shows the mean ± SD of the spleen weights from mice receiving transplants of EV-transduced HSCs (n = 6) or of MLL-AF10/K-ras^G12V co-transduced HSCs (n = 6). ** represents p<0.01.</p